A great deal of evidence now supports the hypothesis that genetically programmed demethylation of 5-methylcytosine (m5C) residues and methylation of cytosine residues in DNA help control development in vertebrates. We have recently made the first identification of a vertebrate protein which binds preferentially to m5C-containing DNA sequences in various types of DNA. This protein, which is termed methylated-DNA-binding protein (MDBP), was partially purified from a nuclear fraction of human placenta. In order to elucidate the role of MDBP in vivo, we describe in this proposal experiments designed to purify MDBP to homogeneity, determine its intracellular distribution, analyze its occurrence in various eucaryotes and procaryotes, find an optimal source of cells for the large scale purification of this specific DNA-binding protein, and characterize the DNA-binding assay with respect to the conditions for maximal binding and the rates of association and dissociation of MDBP.DNA complexes. The specificity of MDBP for DNA sequences methylated in one or both strands will be determined by binding assays with several pairs of precisely tailored DNA molecules. It has been proven that MDBP binds preferentially to DNAs enriched in m5C compared to otherwise identical m5C-poor DNAs. The proposed studies will address the question of whether MDBP also possesses sequence-specificity and, if so, the nature of such specificity. The binding of MDBP to m5C-rich nucleosomal DNA and to m5C-poor nucleosomal DNA; to superhelical m5C-rich DNA and to relaxed m5C-rich DNA; to m5C-containing Z-DNA sequences and to m5C-containing B-DNA sequences will be compared on pairs of analogous ligands. If time permits, studies of the effects of MDBP on in vitro transcription systems and attempts to clone a cDNA for MDBP will be begun. It is proposed that MDBP binds specifically to methylated DNA sequences in vivo as it does in vitro and acts either as a regulatory protein directly controlling the template activity of DNA or as a structural protein altering the local conformation of chromosomes. In either case, MDBP could be a pivotal protein for controlling certain aspects of differentiation and for repressing genes involved in oncogenic transformation or tumor progression.